The human APO-1 (APT) antigen maps to 10q23, a region that is syntenic with mouse chromosome 19.

The APO-1 (APT) antigen is a cell surface antigen expressed on a variety of normal and malignant cells. Binding of anti-APO-1 antibody to the APO-1 antigen induces programmed cell death (apoptosis). The APO-1 antigen shows homology to the members of the tumor necrosis factor receptor/nerve growth factor receptor superfamily. Using cosmid DNA containing the APO-1 gene as a probe for fluorescence in situ hybridization, we have mapped the gene to a subregion of chromosomal band 10q23. The human APO-1 locus lies within a conserved synteny segment present on mouse chromosome 19 consistent with the previous chromosomal assignment of the corresponding mouse antigen.     

Is There a Third Photoreceptor Involved in the Control of Chloroplast Movements in Mougeotia?

The photometric method was used to test a possibility proposed recently that a new photoreceptor with maximum activity at 620 nm is involved in mediating chloroplast rotation in Mougeotia (Z Lechowski, J Bialczyk [1988] Plant Physiol 88: 189-193). The hypothesis was tested under conditions of continuous dichromatic unilateral or mutually perpendicular irradiation with red light of wavelengths 620 or 660 (680) nanometers and far-red. When the red light was polarized parallel to the long cell axis, chloroplast response could be monitored by changing the direction of far-red irradiation. The level of the response obtained with red and far-red applied from the same direction depended on far-red intensity: at higher fluence rates the maximum response was shifted to longer wavelengths of red light. A high fluence rate of far-red inhibited the response. The absorption coefficients of Mougeotia chloroplasts were measured for the studied wave-lengths using the microphotometric method. Possible impact of absorption by the chloroplast on photoreception has been discussed. Current and previous results can be interpreted in terms of phytochrome action and do not support the involvement of the hypothetical 620 nanometer photoreceptor.     

A protein binds the selenocysteine insertion element in the 3'-UTR of mammalian selenoprotein mRNAs.

Several gene products are involved in co-translational insertion of selenocysteine by the tRNA(Sec). In addition, a stem-loop structure in the mRNAs coding for selenoproteins is essential to mediate the selection of the proper selenocysteine UGA codon. Interestingly, in eukaryotic selenoprotein mRNAs, this stem-loop structure, the selenocysteine insertion sequence (SECIS) element, resides in the 3'-untranslated region, far downstream of the UGA codon. In view of unravelling the underlying complex mechanism, we have attempted to detect RNA-binding proteins with specificity for the SECIS element. Using mobility shift assays, we could show that a protein, present in different types of mammalian cell extracts, possesses the capacity of binding the SECIS element of the selenoprotein glutathione peroxidase (GPx) mRNA. We have termed this protein SBP, for Secis Binding Protein. Competition experiments attested that the binding is highly specific and UV cross-linking indicated that the protein has an apparent molecular weight in the range of 60-65 kDa. Finally, some data suggest that the SECIS elements in the mRNAs of GPx and another selenoprotein, type I iodothyronine 5' deiodinase, recognize the same SBP protein. This constitutes the first report of the existence of a 3' UTR binding protein possibly involved in the eukaryotic selenocysteine insertion mechanism.     

Characterization of heat-shock response of the marine bacterium Vibrio harveyi

We have investigated heat-shock response in a marine bacterium Vibrio harveyi. We have found that 39 C was the highest tempature at which V. harveyi was able to grow steadily. A shift from 30° C to 39° C caused increased synthesis of at least 10 proteins, as judged by SDS-PAGE, with molecular masses of 90, 70, 58, 41, 31, 27, 22, 15, 14.5 and 14kDa. The 70, 58, 41 and 14.5 kDa proteins were immunologically homologous to DnaK, GroEL, DnaJ and GroES heat-shock proteins of Escherichia coli, respectively. V. harveyi GroES protein had a lower molecular mass (14.5 kDa) than E. coli GroES, migrating in SDS-PAGE as 15 kDa protein. We showed that a protein of ～43 kDa, immunologically reactive with antiserum against E. coli sigma 32 subunit (σ³²) of RNA polymerase, was induced by heat-shock and co-purified with V. harveyi RNA polymerase. These results suggest that the 43 kDa protein is a heat-shock sigma protein of V. harveyi. Preparation containing the V. harveyi sigma 32 homologue, supplemented with core RNA polymerase of E. coli, was able to transcribe heat-shock promoters of E. coli in vitro.     

Development of a human three-dimensional organotypic skin-melanoma spheroid model for in vitro drug testing

Despite remarkable efforts, metastatic melanoma (MM) still presents with significant mortality. Recently, mono-chemotherapies are increasingly replenished by more cancer-specific combination therapies involving death ligands and drugs interfering with cell signaling. Still, MM remains a fatal disease because tumors rapidly develop resistance to novel therapies thereby regaining tumorigenic capacity. Although genetically engineered mouse models for MM have been developed, at present no model is available that reliably mimics the human disease and is suitable for studying mechanisms of therapeutic obstacles including cell death resistance. To improve the increasing requests on new therapeutic alternatives, reliable human screening models are demanded that translate the findings from basic cellular research into clinical applications. By developing an organotypic full skin equivalent, harboring melanoma tumor spheroids of defined sizes we have invented a cell-based model that recapitulates both the 3D organization and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention. By extending our previous findings on melanoma cell sensitization toward TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) by co-application of sublethal doses of ultraviolet-B radiation (UVB) or cisplatin, we show significant differences in the therapeutical outcome to exist between regular two-dimensional (2D) and complex in vivo-like 3D models. Of note, while both treatment combinations killed the same cancer cell lines in 2D culture, skin equivalent-embedded melanoma spheroids are potently killed by TRAIL+cisplatin treatment but remain almost unaffected by the TRAIL+UVB combination. Consequently, we have established an organotypic human skin-melanoma model that will facilitate efforts to improve therapeutic outcomes for malignant melanoma by providing a platform for the investigation of cytotoxic treatments and tailored therapies in a more physiological setting.     

Arthropod toxins inhibiting Ca2+ and Na+ channels prevent AC‐1001 H3 peptide‐induced apoptosis

Peptide toxins of arthropods are one of the potential sources of bioactive substances. Toxins are able to bind to calcium channels and block them. Ca²⁺ ions play an important role in many cell processes, in particular, in apoptosis. In this work, we study the effect of some arthropod toxins on intracellular processes associated with the induction of apoptosis. Synthetic analogs of U₅‐scytotoxin‐Sth1a, ω‐hexatoxin‐Hv1a, ω‐theraphotoxin‐Hhn2a, and μ‐agatoxin‐Aa1a toxins—inhibitors of calcium L, P, and Q channels and sodium channels were used in the study. Apoptosis was induced by AC‐1001 H3 peptide. We study the effect of toxins on the level of apoptosis, ROS, mitochondrial potential, GSH, and ATP in CHO‐K1 cells. We show that all the tested toxins are able to dose dependently block the induction of apoptosis triggered by AC‐1001 H3 and reduce the level of natural apoptosis in CHO‐K1 cells. Cell incubation with apoptosis inducer AC‐1001 H3 in the presence and absence of toxins causes an increase in the intracellular concentrations of ROS, ATP, and mitochondrial potential and decreases the GSH concentration. The present study reveals the antiapoptotic effect of a number of arthropod peptide toxins. The toxins studied can represent a novel approach used in the treatment of pathologies associated with the activation of apoptotic mechanisms.